Qrt pcr

Trizol Reagent (Invitrogen) was utilized for total RNA extraction from BUMPT cells, HK-2 cells, and kidneys of C57BL/6 J mice. Next, approximately 40 ng of total RNA was reverse transcribed to cDNA using the Prime-Script-RT-Reagent-Kit and the gDNA-Eraser-Kit (RR047A; TaKaRa, Japan). qRT-PCR and SYBR Green (AG11728; Accurate Biotechnology (HUNAN) CO., LTD, ChangSha China) were employed to detect the expression levels of circRNA, micoRNA, and target gene mRNA. Relative quantification was performed by Roche LC 480 determination of ΔCt values (F. Hoffmann-La Roche, Ltd.).
Primers:
Circ_26986 (F: GAACGAACTGCACTCCGCTCTC, R: GCTGCTGGCTTGTCTTGATGATTG)
miRNA-29b-1-5P (F: GCACCGTGCTGGTTTCATATGG, R: ATCCAGTGCAGGGTCCGAGG
RT primer: ATCCAGTGCAGGGTCCGAGG)
hsa_Circ_0072463 (F: TTCCGATGACCAGTTACACAA, R: TTGGTAGTAGCGGCTCCAGT)
PAK7 (F: CTGGGAGAGGTTTGGGAGGAGAG, R: AGGGAACTACTACGGCTGGGAAG)
U6 (F: AGAGAAGATTAGCATGGCCCCTG, R: CAGTGCAGGGTCCGAGGT)
Endogenous Reference Human (F: CCTGGCACCCAGCACAAT, R: GGGCCGGACTCGTCATA)
Endogenous Reference Mouse (F: GGCTGTATTCCCCTCCATCG, R: CCAGTTGGTAACAATGCCATGT)

The total RNA of each sample was extracted from the liver according to the instruction manual of the Trizol Reagent (Beyotime, Nantong, China). The gene sequences of TLR4, NF-κB, and MyD88 in mice were searched on NCBI, and primers were synthesized by Accurate Biology. Table 3 shows the primer sequences. Reverse transcription and qRT-PCR were performed in strict accordance with the manufacturer’s instructions (Accurate Biology, Changsha, China).